I read this paper in which cortisol levels in fish muscle were measured by HPLC-ELISA.
Silva, P. I. M., Martins, C. I. M., Khan, U. W., Gjøen, H. M., Øverli, Ø., & Höglund, E. (2015). Stress and fear responses in the teleost pallium. Physiology & Behavior, 141, 17–22. http://doi.org/10.1016/j.physbeh.2014.12.020
They measured muscle cortisol levels in the following methods;
I would like to know why they measured cortisol levels using ELISA after purification (separation) by HPLC. Why didn't they use HPLC for the quantitation of cortisol level?
If you have any idea about this, kindly let me know. If it is possible to measure the cortisol levels by HPLC-UV without ELISA, I would like to do it.
Hi Yuki, I think the best way to know is by emailing the authors of the particular paper. Meanwhile, Dr. Ton So Ha and Prof. Khalid's lab (Monash Malaysia, school of Biosciences and Jeffrey Cheah School of Medicine) does a lot of HPLC quantification of enzyme / end products such as cortisol and corticosterone. Perhaps you should check with them or their students for the protocol. Normally we will set up a series of standard of known compound, construct a linear graph and get the best-fit line equation. Hope it helps :).
Thank you, Jasmine.
Yes, that's the best way..
I thought I can measure the cortisol levels only with HPLC (not HPLC-ELISA) by making calibration curve using standard solution of different concentrations.
Perhaps there are any reasons they didn't measure the cortisol levels with HPLC-UV itself. (Because their samples were from fish muscle??)
Alright. I need to consult a specialist or someone having enough experience.
Anyway, thank you Jasmine.
You're welcome. Do you mind to share the answer once the authors of the paper reply to your question? I am curious to know too. Thanks and good luck =)
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